Analysis of replicating DNA by velocity sedimentation in sucrose gradients suggested that FV 3 DNA replication occurs in two stages. In the first stage (up to 3 hr p.i.), progeny DNA is synthesized in unit or less than unit length molecules. In the second stage (after 3 hr p.i.), progeny DNA is synthesized in large concatemeric structures which in turn act as precursors for the production of mature viral DNA. Preliminary experiments suggest that first stage DNA replication is restricted to the nucleus, whereas second stage synthesis occurs in the cytoplasm only. In the coming year we would further substantiate these results by (1) biochemical analysis of replicating DNA in purified nuclear and cytoplasmic fractions, and by (2) screening and characterizing the temperature-sensitive mutants that are locked in the first stage of DNA replication at nonpermissive temperature. Such strategy of DNA replication, if confirmed, is strikingly different from any known DNA virus and may represent a transitional stage in the evolution of nuclear and cytoplasmic DNA viruses.